Background: The Repeat Expansion Diseases are caused by intergenerational expansions of a specific tandem repeat. More than 20 such diseases have been identified thus far. The Fragile X-related disorders (FXDs) arise from expansion of a CGG.CCG-repeat in the 5' UTR of the X-linked FMR1 gene. The FXDs are a group of 3 different disorders: Carriers of alleles with 55-200 repeats, so-called premutation (PM) alleles, are at risk for a neurodegenerative disorder, Fragile X-associated tremor-ataxia syndrome and a form of ovarian dysfunction known as FX-associated primary ovarian insufficiency. Furthermore, in females, the premutation allele can undergo expansion on intergenerational transfer that can result in their children having alleles with >200 repeats. This expanded allele is known as a full mutation and individuals who inherit such alleles almost always have Fragile X syndrome (FXS), which is the leading heritable cause of intellectual disability. The mechanism by which is expansion occurs is unknown. It is thought to differ from the generalized microsatellite instability seen in many different cancers in that the instability is confined to a single genetic locus and shows a strong expansion bias. Expanded alleles are also associated with a folate-sensitive fragile site that is coincident with the repeat on the X chromosome. This site, which gives the disorder its name, is one of many fragile sites present on the human genome. These sites are prone to breakage and in some cases are associated with deleterious chromosome deletions and translocations. In the case of the FX fragile site, there is reason to believe that it is responsible for the high frequency loss of the affected chromosome resulting in Turner syndrome (45, X0) in female carriers of a FM allele. Progress report: We have shown previously that the mismatch repair protein MSH2 is absolutely required for all intergenerational and somatic expansions in a knockin mouse model for these disorders (Lokanga, Zhao and Usdin, 2014). We also showed that expansion occurs independently of chromosomal duplication in a process that is dependent on transcription or the presence of the predisposed allele in a transcriptionally competent region of the genome (Lokanga, Zhao, Entezam and Usdin, 2014). However, this requirement for transcriptional competence does not reflect a role for Transcription Coupled Repair (TCR), a form of nucleotide excision repair that is confined to actively transcribed genes (Zhao and Usdin, 2014). In fact we have shown that although CSB, a protein that is essential for TCR, plays an auxiliary role in promoting expansions (Zhao and Usdin, 2014) it also protects the genome against expansions, presumably via its participation in another DNA repair pathway (Zhao and Usdin, 2015). In the last reporting period we also showed that a hypomorphic mutation in Pol , a key polymerase involved in Base Excision Repair (BER), reduces the expansion frequency. This implicates central events in BER in the expansion process. Since BER is the major pathway by which oxidative damage to DNA is repaired in mammalian cells, this observation is consistent with our earlier observation that oxidative stress increases expansion risk (Entezam et al., 2010). In this reporting period we have extended our studies on mismatch repair proteins involved in repeat expansion. We showed that loss of MSH3, the binding partner of MSH2 in the MutSbeta complex, results in the loss of all but 2% of intergenerational expansions and most if not all somatic expansions (Zhao et. al., 2015). This suggests that MutSbeta is required for almost all expansions with the residual expansions seen in Msh3 null mice likely representing the ability of MutSalpha to substitute, albeit poorly for MutSbeta in the expansion process. We also showed that in mice lacking MSH6, the MSH2 binding partner in the MutSalpha complex, that the expansion frequency was reduced by much more than the 2% we expected from the effect of the loss of MSH3. This somewhat surprising observation suggests that while MutSalpha is not able to substitute very effectively for the loss of MutSbeta, it does play an unexpectedly large role in promoting expansions in the FX mouse model. Our data also raise the possibility that it could contribute to expansions in other repeat expansion diseases as well. In vitro experiments with purified proteins suggest that one way the MutSalpha may be acting is via the stabilization of the hairpins that are thought to be the substrates for expansion. MutSalpha also facilitates the binding of MutSbeta to the FX hairpins. Thus our data demonstrates that expansion involves an unusual nexus of 3 sets of DNA repair proteins that normally act to protect the genome against mutation, but that in the context of the FMR1 locus are actually involved in the generation of the mutation that causes the FXDs.